P702: APPLICATION OF NEXT GENERATION SEQUENCING (NGS) FOR MINIMAL RESIDUAL DISEASE (MRD) AND CHIMERISM MONITORING IN MYELODYSPLASTIC SYNDROME (MDS) AFTER ALLOGENIC STEM CELL TRANSPLANTATION (ASCT)
Authors: Irene Zamanillo, Santiago Barrio, Alejandro Martin, Sara Dorado, Yanira Heredia, Margarita Rodriguez, Inmaculada Rapado, Ana Isabel Jimenez Ubieto, Rosa Ayala Diaz, Joaquín Martinez-Lopez, Maria Teresa Cedena Romero
Journal: HemaSphere (2023)

Background: Next Generation Sequencing (NGS) is a rapidly evolving technique that simultaneously identifies several somatic mutations and quantifies allele burden. Therefore, it could be suitable for MRD monitoring in MDS patients. Traditionally its main limitations have been high single base error rates, which limited sensibility to 0.1-1%. However, sensitivities up to 10-4 can be achieved by applying novel ultrasensitive personalized sequencing techniques. Aims: This study explores the applicability of NGS for MRD and chimerism monitoring in MDS patients in the post-transplant setting. Methods: Twenty patients underwent ASCT between 2017 and 2022 in our center. Seven patients were excluded due to procedure-related mortality or relapse before 100-day follow-up, and six were excluded due to unavailable samples. We finally sequenced twenty-five samples from seven patients. Donor and receptor genomic DNA was sequenced with an in-house PhasedSNP panel that covers 58 regions with paired polymorphisms in less than 15bp. Receptor DNA was also sequenced with a 42-gene NGS panel (Ion TorrentTM) to identify somatic mutations used in follow-up samples to quantify MRD as previously described (Onecha E. Haematologica 2019). Briefly, an amplicon-multiplexed mini-panel was defined for every patient to detect the selected MRD biomarkers and donor/receptor phased SNPs identified at diagnosis. The mini-panel included molecular-tagged primer pairs to amplify every selected marker in three biological replicates, finally combined in the same library. Final libraries were sequenced on the Ion S5 System platform with an estimated depth of 500,000× per amplicon (median 151,242 reads). A median of 648 ng (range 180 - 662.4 ng) from bone marrow follow-up samples was sequenced. Results: The mean age at diagnosis was 53.6 years (SD 8.6). Risk at diagnosis according to IPSS-R score was intermediate in one patient (14.2%), high in four (57.1%) and very high in two (28.5%). The NGS-based MRD testing demonstrated high applicability: 6/7 patients (85.7%) had at least one somatic mutation suitable for MRD monitoring and 5/7 patients had more than one mutation. Phased SNPs chimerism was applicable in all the subjects. In most patients, we detected low levels of both MRD and receptor chimerism in post-transplant samples, usually below 1%. For all post-ASCT samples, MRD detection using flow cytometry was negative. The correlation between phased SNPs chimerism and MRD detection, both NGS-based, was very high (R=0.922; p=2.85 · 10-9) (Figure 1). However, qPCR-based chimerism and NGS-based phased SNPs chimerism showed some differences: in five cases, NGS-chimerism levels below 0.5% were undetectable by qPCR. NGS detected all cases with positive qPCR chimerism, but in six samples, chimerism quantification was slightly different between the two techniques. Summary/Conclusion: NGS-based MRD detection in MDS is feasible and has high applicability and sensitivity up to 10-4, with several advantages compared to traditional methods. Further research is warranted to study the relationship between MRD detection and clinical outcomes.Keywords: Myelodysplastic syndrome, Measurable residual disease, Chimerism quantification